The present invention relates to ephrin reverse signaling in vertebrate cells, particularly cerebellar granular cells and leukocytes, the signaling acting through a novel PDZ-RGS protein, to block a heterotrimeric G protein-pathway. This signaling results in inhibition of the chemoattractant effects of a chemokine, in particular, of SDF-1. Methods and compositions for modulation of the pathway provide potential therapeutic agents for inflammation and autoimmune diseases.
Chemoattractant cytokines or chemokines are a family of proinflammatory mediators that promote recruitment and activation of multiple lineages of leukocytes and lymphocytes. They can be released by many kinds of tissue cells after activation. Continuous release of chemokines at sites of inflammation mediates the ongoing migration of effector cells in chronic inflammation. The chemokines characterized to date are related in primary structure. They share four conserved cysteines, which form disulfide bonds. Based upon this conserved cysteine motif, the family is divided into two main branches, designated as the C—X—C chemokines (α-chemokines), and the C—C chemokines (β-chemokines), in which the first two conserved cysteines are separated by an intervening residue, or adjacent respectively (Baggiolini, M. and Dahinden, C. A., Immunology Today, 15:127-133 (1994)).
The C—X—C chemokines include a number of potent chemoattractants and activators of neutrophils, such as interleukin 8 (IL-8), PF4 and neutrophil-activating peptide-2 (NAP-2). The C—C chemokines include RANTES (Regulated on Activation, Normal T Expressed and Secreted), the macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β), and human monocyte chemotatic proteins 1-3 (MCP-1, MCP-2, MCP-3), which have been characterized as chemoattractants and activators of monocytes or lymphocytes but do not appear to he chemoattractants for neutrophils. Chemokines, such as RANTES and MIP-1α, have been implicated in a wide range of human acute and chronic inflammatory diseases including respiratory diseases such as asthma and allergic disorders.
The chemokine receptors are members of a superfamily of G protein-coupled receptors (GPCR) which share structural features that reflect a common mechanism of action of signal transduction (Gerard, C. and Gerard, N. P., Annu Rev. Immunol., 12:775-808 (1994); Gerard, C. and Gerard, N. P., Curr. Opin. Immunol., 6:140-145 (1994)). Conserved features include seven hydrophobic domains spanning the plasma membrane, which are connected by hydrophilic extracellular and intracellular loops. The majority of the primary sequence homology occurs in the hydrophobic transmembrane regions with the hydrophilic regions being more diverse.
The superfamily of GPCRs has at least 250 members (Strader et al. FASEB J., 9:745-754, 1995; Straderet al. Annu. Rev. Biochem., 63:101-32, 1994). It has been estimated that one percent of human genes may encode GPCRs. GPCRs bind to a wide-variety of ligands ranging from photons, small biogenic amines (i.e., epinephrine and histamine), peptides (i.e., IL-8), to large glycoprotein hormones (i.e., parathyroid hormone). Upon ligand binding, GPCRs regulate intracellular signaling pathways by activating guanine nucleotide-binding proteins (G proteins). GPCRs play important roles in diverse cellular processes including cell proliferation and differentiation, leukocyte migration in response to inflammation, and cellular response to light, odorants, neurotransmitters and hormones (Strader et al., supra.).
Over the last fifteen years it has become apparent that many ligands that signal through cell surface receptors are themselves transmembrane molecules (Pfeffer and Ullrich, 1985; Flanagan et al., 1991; Massague and Pandiella, 1993). One function of this ligand anchorage may be to tightly localize the signal. This idea is particularly well exemplified by the ephrins, since they require membrane anchorage to activate their receptors in a direct cell-cell contact mechanism, and since they have spatially precise patterning roles.
A second potential function for transmembrane ligands is to allow bi-directional signaling. Again, the ephrins have provided a particularly good model system to investigate this idea. Reverse signaling through B ephrins has been demonstrated biochemically by ligand phosphorylation. Evidence of important developmental roles has come from genetic and embryological studies of whole embryos or tissues.
Ligands in the ephrin-B family are cell surface anchored by a transmembrane domain, and signal through their Eph receptors by direct cell-cell contact (Davis et al., 1994; Drescher et al., 1997; Flanagan and Vanderhaeghen, 1998; Frisen et al., 1999; Holder and Klein, 1999; Mellitzer et al., 1999). This contact-mediated mechanism provides the potential for bi-directional signaling, with a forward signal through the tyrosine kinase receptor, and a reverse signal through the ligand. Reverse signaling has been demonstrated biochemically by studies showing B ephrins become phosphorylated upon treatment of cells with soluble EphB-Fc receptor fusion protein (Holland et al., 1996; Bruckner et al., 1997). In the context of whole organisms or tissues, genetic and embryological studies have supported important roles for B ephrin reverse signaling in developmental processes, including axon pathway selection, blood vessel formation, and rhombomere compartmentation (Henkemeyer et al., 1996; Jones et al., 1998; Wang et al., 1998; Adams etal., 1999; Gerety etal., 1999; Mellitzer et al., 1999:Xu et al., 1999). However, little is known of the specific effects of B ephrin reverse signaling on individual cells, or the signal transduction pathways that lead to such effects.
Evidence that B ephrins might interact with cytoplasmic proteins initially came from sequence comparison of ephrin-B1 and -B2, which show a striking 100% amino acid identity in the last 33 amino acids of the intracellular domain (Bennett et al., 1995; Bergemann et al., 1995). Using the intracellular domain in yeast two-hybrid screens, several binding proteins have been identified (Torres et al., 1998; Bruckner et al., 1999; Lin et al., 1999). All the binding proteins identified to date contain a PDZ (PSD-95/Dlg/ZO-1) domain, a protein module that binds the C-termini of membrane proteins. PDZ proteins have been widely implicated in forming sub-membrane scaffolds that cluster molecules at the cell surface (Craven and Bredt, 1998; Garner et al., 2000; Sheng and Pak, 2000).
RGS proteins form a large molecular family identified in recent years, with more than 20 members in mammals (Arshavsky and Pugh, 1998; Kehrl, 1998; De Vries and Farquhar, 1999; Zheng et al., 1999). They act as GTPase activating proteins (GAPS) for heterotrimeric G proteins, accelerating the G protein catalytic cycle and thereby facilitating rapid signaling processes such as retinal phototransduction (Arshavsky and Pugh, 1998). Many RGS proteins contain additional motifs, including PDZ domains, leading to suggestions that they could couple G proteins with other signaling pathways (Kehrl, 1998; De Vries and Farquhar, 1999). The RGS protein pl15RhoGEF has separate domains that regulate both heterotrimeric and small G proteins, while nematode EAT-16 mediates a genetic interaction between two heterotrimeric G protein pathways (Hart et al., 1998; Kozasa et al., 1998; Hajdu-Cronin et al., 1999). However, there is generally little functional evidence on the specific significance of combining RGS domains with other domains, including a potential role for PDZ-RGS proteins in regulating G proteins in response to extracellular signals.
Heterotrimeric G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate the effects of many extracellular signals (Watson and Arkinstall, 1994; Bargmann and Kaplan, 1998). Some of the best characterized guidance molecules act through GPCRs (Parent and Devreotes, 1999), notably the chemokines, which are leukocyte chemoattractants with important roles in immunity (Melchers et al., 1999). A role for chemokines in neural development was shown more recently. The radial movement of cerebellar granule cells is a well characterized model for neural migration (Rakic, 1990; Hatten, 1999) and occurs prematurely in mice with gene disruptions of the chemokine SDF-1, or its receptor CXCR4 (Ma et al., 1998; Zou et al., 1998). Heterotrimeric G protein signaling may also mediate, at least in part, the actions of Netrins, Semaphorins and other neural guidance molecules, though these pathways are generally less well understood (Vancura and Jay, 1998; Corset et al., 2000; Nakamura et al., 2000).